J. Phys. Chem. B 1999, 103, 6605-6610
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Rate Constants and Free-Energy Plot for the Reduction of Horse-Heart Cytochrome c(III) by Eight Organic Radicals Christopher D. Borman,† A. Mark Dobbing,† G. Arthur Salmon,‡ and A. Geoffrey Sykes*,† Department of Chemistry, The UniVersity of Newcastle, Newcastle upon Tyne, NE1 7RU, U.K., and Cookridge Radiation Research Centre, The UniVersity of Leeds, Cookridge Hospital, Leeds LS16 6QB, U.K. ReceiVed: NoVember 23, 1998
Rate constants k12/M-1 s-1 (22 °C) for the reduction of the oxidised form of horse-heart cytochrome c by eight organic radicals (OR), e.g., methyl viologen MV•+, determined by pulse radiolysis and/or stopped-flow methods, correlate well in a plot of log k12 vs reduction potential E°1 for the OR. In the pulse radiolysis experiments the ORs were generated in situ by rapid reduction of the parent form by the formate radical CO2•-, and in the stopped-flow experiments prior reduction of the parent was with dithionite. In both procedures it was necessary to consider formation of double-reduced parent forms. Rate constants k12 are in the range 109 to 104 and E°1 values from -0.446 to +0.194V. The Marcus free-energy plot of log10 k12 - 0.5 log10 f vs E°1/0.059 gives a slope of 0.49, in excellent agreement with the theoretical value of 0.50, with an intercept at -E°1/0.059 ) 0 of 6.55 in good agreement with a calculated value of 6.51 from known parameters. The latter includes the reduction potential E°2 (0.263V) and self-exchange rate constant k22 (0.36 × 103 M-1 s-1) for the cytochrome c(III)/(II) couple, and assumes a common self-exchange rate constant k11 of 1.0 × 106 M-1 s-1 for the ORs.
Introduction Cytochrome c is a small structurally well characterised protein containing an Fe-porphyrin prosthetic group (Mr 12.4 kDa; 104 amino acids), which is a component of the mitochondrial electron-transport chain.1 The aim in the present studies was to determine rate constants (k12) for cross reactions of cytochrome c(III) with organic radicals (OR), e.g., MV•+ (1),
MV•+ + cyt c(III) f MV2+ + cyt c(III)
(1)
and to see whether the k12 values conform to the Marcus relationship (2),2
k122 ) k11k22K12f
(2)
assuming a single common self-exchange rate constant (k11) for the ORs, with K12 the equilibrium constant for the cross reaction and k22 the self-exchange rate constant for the cytochrome c(III)/ (II) couple. The term f is defined as in (3)
log f )
(log K12)2 4 log (k11k22/Z2)
(3)
where the collision frequency Z is assumed to be 1011 M-1 s-1. Using the Nernst equation, log K12 ) (E°2 - E°1)/0.059, with E°1 and E°2 reduction potentials in volts (vs NHE) for the parent/ OR and cytochrome c(III)/(II) couples, respectively, (2) can be expressed as in (4). †
Department of Chemistry. ‡ Cookridge Radiation Research Centre.
(log k12 - 0.50log f) ) 0.50(log k11 + log k22 + E°2/0.059) - 0.50 E°1/0.059 (4) This was tested by plotting the left-hand side of (4) against -E°1/0.059, for the eight ORs. Reduction potentials E°1 for the ORs cover a wide range (-0.446 to + 0.194 V) appropriate for reduction of cytochrome c(III), E°2 ) 0.263V.3 Electron-transfer reactions of cytochrome c have been widely studied1,4,5 and in many cases are believed to occur at the solvent accessible edge of the porphyrin.6,7 A preference is shown by physiological partners for reaction at an area centred around the point at which the positive end of the dipole axis of cytochrome c crosses the surface of the protein. This region is near to the β-carbon of the Phe-82 (midway between Lys-13 and Lys-72) and incorporates part of the porphyrin exposed edge.1,6 Reactions of metalloproteins with free radicals have not previously been studied in a systematic manner, and we set out therefore to explore the behavior of eight such radicals with a well characterized protein. The faster reactions were studied by pulse radiolysis (PR),8 with the formate radical CO2•- (E° ) -1.9V) used to generate the ORs in situ. The slower reactions were studied using the stopped-flow method (SF), with dithionite used to reduce the parent OR’s in a prior step. For reactions which give a linear plot of (4) the slope should be equal to 0.50 and the intercept to 0.50 (log k11 + log k12 + E2°/0.059). Related questions are whether the ORs react at the same site on cytochrome c, have a common self-exchange rate constant k11,9-11 and whether the charge on the OR (1 + to 2 -) is important. Experimental Section Protein. Horse-heart cytochrome c from Sigma Chemicals was purified as cytochrome c(III) by a procedure already described.12 Fractions with UV-Vis absorbance (A) ratios A550
10.1021/jp984520g CCC: $18.00 © 1999 American Chemical Society Published on Web 07/21/1999
6606 J. Phys. Chem. B, Vol. 103, No. 31, 1999
Borman et al.
Figure 1. Formulas of eight parent reagents (X) used to generate organic radicals (X•-) used in this study.
(reduced) to A280 (oxidized) > 1.15 were used for the kinetic studies described. Concentrations were determined from the absorbance at 416 nm (1.29 × 105 M-1 cm-1) for reduced protein, with ∆ (reduced > oxidized) of 4.0 × 104 M-1 cm-1 at 416 nm.1,13 Reagents for Organic Radicals. The following parent reagents (Figure 1), with trivial, systematic, and abbreviated names as indicated, were used in this work: methyl viologen, C12H14N2Cl2 (1,1′-dimethyl-4,4′-bipyridinium dichloride), MV2+; benzyl viologen, C24H22N2Cl2, (1,1′-dibenzyl-4,4′-bipyridinium dichloride), BV2+; phenosafranin, C18H15N4Cl (3,7-diamino-5phenylphenazinium chloride), Pf+; riboflavin C17H20N4O6 (vitamin B2), Rb; Resorufin, C12H6NO3Na, (7-hydroxy-3Hphenoxazine-3-one sodium salt), Rf-; methylene blue, C16H18N3SCl (3,7-bis(dimethylamino)phenazothionium chloride), MB+; toluidine blue (C15H6N3SCl)2ZnCl2 (approximate formula only), TB+; and indophenol C12H6Cl2NO2Na (phenolindo-2,6-dichlorophenol sodium salt), IP-. These were obtained from Sigma Chemicals, except MB+ and IP- (BDH), and TB+ (Fluka). Toluidine blue (1 g) was recrystallized to constant UV-Vis spectrum and reactivity by first dissolving in H2O (18 mL) at 80 °C. Ethanol (13.5 mL) was added with stirring over ∼10 min at 0 °C. The solid obtained was filtered off, washed with cold ether, and stored in a desiccator over silica gel (yield 28%). Titration of the organic redox component with dithionite gave 81% reactivity, as required by the formula (C15H6N3SCl)2ZnCl2 indicated. Some difficulties were encountered in dissolving resorufin to the required levels, and stock solutions were prepared by sonicating for 1 h and then filtering through a 2 µm Acrodisc filter (Gellman). Identical UV-Vis spectra were obtained by making up more dilute solutions by weight, which also gave relevant absorption coefficients (). All experiments involving ORs were carried out under rigorous O2-free conditions using 99.9% N2 gas. Electrochemistry. Reduction potentials (E°1 vs NHE) for the 1-equiv parent/OR couples were checked by cyclic voltammetry (CV) using a Princeton Applied Research model 173 potentiostat. Control of the equipment was by interfacing to an IBM compatible PC with software from EG & G. A three-electrode system consisting of working (glassy carbon or gold), auxiliary (platinum wire), and reference (Ag/AgCl) electrodes was used. The glassy carbon (or gold) electrode was polished using an
Al2O3 slurry (0.30 µm and then 0.015 µm particle size) and Buehler polishing cloth, rinsed with deionized H2O, and then sonicated in H2O for ∼5 min to remove all traces of Al2O3 (which can affect readings). Polishing was carried out prior to each scan. The cell was calibrated using the [Fe(CN)6]3-/4couple (0.410 V vs NHE in 0.10 M NaCl). Concentrations of OR parent used were in the 0.1-10.0 mM range, with pH 7.0 (45 mM phosphate), I ) 0.100M, and the temperature ∼20 °C. Measured E° values from CVs were converted to E°1 values vs normal hydrogen electrode (NHE) by adding 0.222 V to the value determined vs Ag/AgCl. With two exceptions E°1 values were within 10 mV of the literature values,14 see values included in Table 4 below. One exception is with toluidine blue, which gave a value 0.015 mV as compared to a literature value of 0.034 V.15 With indophenol16 a 23 mV smaller value was observed. In the case of methylene blue it was necessary to use concentrations at the lower end of the range (0.10 mM) to obtain symmetrical cyclic voltammograms. Under these conditions a value E°1 of 0.004 V was obtained, as compared to a literature value of 0.011 V.14 Formation of the double-reduced form probably accounts for the unsymmetrical voltammograms.17,18 Procedure for Pulse Radiolysis. Experiments were carried out on a van de Graaff accelerator at the Cookridge Radiation Research Centre, University of Leeds, using a triple-pass cell (6.9 cm light pathlength) and a 2.5 MeV (∼4 × 10-16 J) beam of electrons.11 Pulse lengths were 0.6 µs, and the yield of reducing radicals R for a given pulse is described by (5).
[R] ) Vsec Ssec Gr
(5)
The secondary emission chamber voltage Vsec (units V) was measured directly, and the sensitivity Ssec (units GyV-1) was obtained by standard thiocyanate dosimetry.19 The chemical yield of radicals per joule of energy absorbed by the system is Gr (units mol J-1). Under the conditions adopted the production of radicals by each pulse was as in (6),
4.25H2O f e-aq(2.75), OH‚(2.85), H‚(0.67), H2(0.47), H2O2(0.75) (6) where 107 G values in parentheses correspond to the number
Reduction of Horse-Heart Cytochrome c(III)
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of moles of product per joule of energy absorbed. Solutions contained sodium formate (0.010 M) were buffered with phosphate (40.5 mM) and saturated with N2O, I ) 0.100 M. Subsequent reactions are (7)-(9)
Additional information is available from previous PR studies on methylene blue. Thus the OR MB• absorbs strongly at 420 nm ( ) 1.04 × 104 M-1 cm-1),17 and the equilibrium constant for (12)
e-aq + N2O + H2O f N2 + OH- + OH•
(7)
2MB• a MB+ + MB-
OH•/H• + HCO2- f CO2•- + H2O/H2
(8)
CO2•- + X f CO2 + X•-
(9)
where X•- in the present case is the OR and X the parent form. A large excess of X (1.0 × 10-4 M) was used so that the formate radical CO2•- is effectively scavenged, generating OR (X•-) as the only reducing species present in solution. With such a choice of reactant concentrations double reduction of the parent is precluded, but disproportionation of X•- as in (10),
X•- + X•- a X + X2-
(10)
needs to be considered. Experiments were in the temperature range 22 ( 1 °C. Stoichiometries of Parent Forms with Dithionite. Prior to SF experiments stoichiometries were determined for the reactions of the four colored parent forms Rf-, MB+, TB+, and IP- (∼30 µM) by titration with sodium dithionite Na2S2O4 (Sigma; ∼2 mM) in a glove box (O2 levels < 3 ppm). Dithionite is a 2 equiv reductant (11).
S2O42- - 2e- f 2SO2 (i.e., SO32-)
(11)
Solutions of Na2S2O4 were made up by weight, and the purity determined as 84% by spectrophotometric titration with excess [Fe(CN)6]3- peak at 420 nm ( ) 1010 M-1 cm-1). Reactions were rapid (∼1 min), and the number of moles of S2O42required to bring about bleaching of 1 mole of parent were close to 1.0 for the following: Rf- (0.96), MB+ (0.98), TB+ (0.96). In the case of IP-, which has a quinone type structure, only 0.47 mol were required. Therefore, in the first three cases double reduction of the parent OR is occurring, and the consequences of this need to be considered in the SF experiments. Procedure for Stopped-Flow. An Applied Photophysics UV-Vis stopped-flow spectrophotometer was used to monitor reactions. Rigorous O2-free conditions were achieved by replacing connecting leads on the stopped-flow by polyetheretherketone (PEEK) tubing, which has low O2 permeability, and by prior bubbling of N2 through reactant solutions. The cytochrome c(III) oxidant was present in excess. Reduction of the four parents Rf-, MB+, TB+, and IP- by dropwise addition of dithionite (∼10 mM)20 was carried out using a Gilson pipetteman in the glove box until only a faint color of the parent remained. Excess dithionite was avoided because it is able to reduce cytochrome c(III) directly.21 The stoichiometry experiments have indicated that under the conditions adopted, formation of the double-reduced products of Rf-, MB+, and TB+ occurs and approaches completion. In the case of IP-, however, the singly-reduced product is obtained. Reactions were monitored at the peak positions λ/nm of the parent forms for Rf(570), MB+ (660), TB+ (632), and IP- (603). At wavelengths >600 nm, the /M-1 cm-1 for cytochrome c(III) (
